sequencing - which primer should I trust? (Dec/10/2006 )

Hi everyone,

I met a problem with a sequencing, actually I made a point mutation and when I look at the sequencing I got the right mutation only on the forward primer so I could trust this primer since the chromatogram seems not too bad at the mutation's place but I also got an addition of 4 nucleotides which is not present in the reward primer. I could sequence again but it takes 3 days before getting the results and I wanted to purify the protein right now. What would you do?

-vik-

I would wait for the resequencing results.

How many plasmids isolates are you sequencing? Do you have more then one isolate of this plasmid type? I would actually sequence at least 3 difference isolates of this plasmid as a precaution that one or more plasmids contains a sequence error.

However, if you don't mind burning time (if there is an error), then go ahead and do the protein induction.

-perneseblue-

QUOTE (perneseblue @ Dec 10 2006, 09:38 PM)

I would wait for the resequencing results.

How many plasmids isolates are you sequencing? Do you have more then one isolate of this plasmid type? I would actually sequence at least 3 difference isolates of this plasmid as a precaution that one or more plasmids contains a sequence error.

However, if you don't mind burning time (if there is an error), then go ahead and do the protein induction.

Thanks for your reply. I have used only one isolate plasmid so maybe I should purify an other isolate plasmid which might not contain the addition of nucleotides. Anyway I'm gonna wait for the second sequencing.

C U.

-vik-

I would take 2 or 3 colonies to and do sequencing per duplicate. And I would wait for resequencing too. If not, there is a risk you’ll waste time and reactives!!!

-aztecan princess-

Yes, I agree with others. It's better for you to sequence a few more colonies (another one or two), rather than wasting your time for the protein work if it is the wrong plasmid.